The term chromatography embraces a family of closely related separation methods. The feature distinguishing chromatography from most other physical and chemical methods of separation is that two mutually immiscible phases are brought into contact wherein one phase is stationary and the other mobile. The sample mixture, introduced into the mobile phase, undergoes a series of interactions (partitions) many times before the stationary and mobile phases as it is being carried through the system by the mobile phase. Interactions exploit differences in the physical or chemical properties of the components in the sample. These differences govern the rate of migration of the individual components under the influence of a mobile phase moving through a column containing the stationary phase. Separated components emerge in the order of increasing interaction with the stationary phase. The least retarded component elutes first, the most strongly retained material elutes last. Separation is obtained when one component is retarded sufficiently to prevent overlap with the zone of an adjacent solute as sample components elute from the column.
The column is the heart of the chromatograph and provides versatility in the type of instrument that can be obtained by a single instrument. Especially, large efforts are continuously being made to design the optimal stationary phase for each specific purpose. Such a stationary phase is usually comprised of a support or base matrix to which a ligand comprising functional i.e. binding groups has been attached. As is easily realised, the selection of possible ligands is vast but for an overview a number of different classes of ligands will be given below.
In affinity adsorption, serine proteases have been adsorbed/desorbed to/from matrices to which p-aminobenzamidine has been covalently linked via the para amino group.
Mixed mode anion-exchangers have been disclosed e.g. in WO 9729825 (Amersham Pharmacia Biotech AB), providing interactions based on charges and hydrogen-bonding involving oxygen and amino nitrogen on 2–3 carbons' distance from positively charged amine nitrogen. The publication is based on the discovery that this kind of ligands can give anion-exchangers that require relatively high ionic strengths for eluting bound substances.
Cation-exchangers in which there are mixed mode ligands that require relatively high ionic strengths for eluting bound substances have been suggested in WO 9965607 (Amersham Pharmacia Biotech AB). Furthermore, WO 9729825 (U.S. Pat. No. 6,090,288) and WO 9965607 describe anion and cation-exchange ligands that both require relatively high elution ionic strength.
Separation media of the general structure M-SP1-L, wherein M is a support matrix that may be hydrophilic, SP1 is a spacer and L comprises a mono- or bicyclic homoaromatic or heteroaromatic moiety that may be substituted (a homoaromatic moiety comprises an aromatic ring formed only by carbon atoms) are disclosed in WO 9808603 (Upfront Chromatography). The substituents are primarily acidic. The separation medium is suggested for the adsorption of proteins, in particular immunoglobulins, by hydrophobic interactions rather than ion exchange. WO 9600735, WO 9609116 and U.S. Pat. No. 5,652,348 (Burton et al) also disclose separation media that are based on hydrophobic interaction. Adsorption and desorption are supported by increasing or decreasing, respectively, the salt concentration of the liquid or changing the charge on the ligand and/or the substance to be adsorbed/desorbed by changing pH. The ligands typically comprise a hydrophobic part that may comprise aromatic structure. Some of the ligands may in addition also contain a chargeable structure for permitting alteration of the hydrophobic/hydrophilic balance of the media by a pH change. The chargeable structure may be an amine group.
Finally, U.S. Pat. No. 5,789,578 (Burton et al) suggests to immobilise a thiol containing ligand, such as 3-mercaptopropionic acid, glutathione etc, by addition of the thiol group over carbon-carbon double bond attached to a support matrix.
However, once the structure of the desired ligand has been decided, further important considerations will reside in the choice of a suitable method of preparation thereof.
For example, recently a novel type of ligands denoted high salt ligands has been disclosed, see e.g. WO 0011605 (Amersham Pharmacia Biotech AB, Uppsala, Sweden). Since these ligands can function as mixed mode cation-exchanger ligands, they have shown to be of great interest in many industrial applications, such as protein purification, since they can withstand high salt concentrations and accordingly does not require any substantial dilution of the sample. Thus, the high salt ligands are advantageously used for separations, since they reduce the total volume of sample required as compared to previously described methods, and accordingly reduce the total cost for equipment as well as work effort required in such applications.
However, even though the mixed mode cation-exchanger ligands reduce costs and efforts when used in separation, the hitherto described methods of preparation thereof includes drawbacks that have made them less advantageous in practice. In general terms and to obtain a good diversity, the preparation of such ligand would include four steps, namely                (i) Introduction of a reactive group on a chromatographic support and its optionally activation thereof;        (ii) Reacting the resulting modified support with a thiol compound containing an acid function.        (iii) Activation of the acidic functions of the solid support with a suitable reagent (ex. NHS in presence of DCC) in an organic solvent.        (iv) Addition of an amino acid derivative comprising a suitable residue R to produce a finished ligand attached to a support.        
One problem with the sequence of steps disclosed above is that it will result in a product which in fact contains two different ligands, namely the thio ether linker containing an acid function, resulting from unsuccessful conversions in step (iii) or (iv) and the desired end product. A chromatographic support composed of a mixture of two such different ligands can cause several problems when used in a separation procedure. For example, the analysis will become difficult, resulting in less robust methods of preparation and use than what is generally needed. For the same reason, if the media is obtained as a mixture of two ligands, it will become difficult to optimise the preparation and the use of such chromatographic support for each specific application. Furthermore, such a mixture will inherently result in a less specific separation than a better-defined medium.
Furthermore, another problem involved in the conventional method described above is the fact that the large chromatographic support molecule will be present in the procedure from step (i). Put differently, the whole procedure will need to be performed in large volumes, requiring very specific large-scale equipment and entailing the substantial costs that are inherent in working in such type equipment.
Yet another drawback with the prior art method disclosed above is the fact that all steps are performed on solid support. The use of the above-discussed large specific equipment will also include additional disadvantages related to more time-consuming and complicated washing routines. This is especially true for steps (ii) to (iii) and (iii) to (iv), where you go from an aqueous to an organic solvent and vice versa.
Since at present there are no functional alternatives to the method described above available, there is a need within this field of improved methods for the manufacture of mixed mode cation-exchanger ligands for use in separation procedures.